Vitamin d distillation process and product



Patented Apr. 6, 1943 VITAMIN D DISTILLATION PROCESS AND PRODUCT Kenneth C. D. Hickman and Edward Le B. Gray,

Rochester, N. Y.,

assignors to Distillation Products, Inc., Rochester, N. Y., a corporation of Delaware No Drawing. Application April 13, 1939, Serial No. 267,716. In Great Britain April 20, 1938 3 Claims.

This inventionrelates to new distillation procedure and products produced thereby.

An object of the invention is to provide a new distillation process. A further object is to provide products of distillation which have different biological properties from those heretofore prepared. Another object is to provide an improved method for separating a plurality of vitamin D products from fish oils. Another object is to provide new vitamin products. Other objects will become apparent from the following description and claims.

We have found that a plurality of antirachitic substances occur in natural fish oils and that they can be separated therefrom by certain procedure including fractional high vacuum, unobstructed path distillation.

In separating the antirachitic substances we prefer to first saponify the fish oil and separate the non-saponifiable matter from the saponification mixture. This non-saponifiable matter contains the multiple forms of the antirachitic substances in alcohol'form and also impurities such as sterols, etc. We prefer to employ saponification procedure which is conventional in the art for preparing vitamin concentrates by saponification. Sinc this procedure is well known-it does not appear that it would be of value to describe it in further detail.

The reason for the preliminary saponification is that the vitamin D's are present in fish oils mostly in ester form. For instance in the case tillations is spread over a relatively large area, it is diflicult to remove one fraction from the condensing surface before the next fraction has condensed thereon. In other words, it is diflicult conditions and a mixture of the desired fraction of cod-liver oil approximately 70% of the antirachitic substances present are esters. If the esters were not saponified some admixture of the different forms'of vitamin D would probably take place. This is due to the fact that an ester of a low molecular weight fatty acid and a, high molecular weight vitamin D might distill at the same temperature as an ester of a high molecular weight fatty acid and a low molecular weight vitamin D. A poor separation would result in such a situation. Although saponification is indicated as preferred procedure, it should not be concluded that separation of the different forms of vitamin D by direct fractionating of the fish oil is impossible. As a matter of fact, separation can be accomplished in this manner, even though somewhat greater overlapping of the distillation ranges takes place. We, therefore, contemplate sucsh, procedure as being within the scope of my invention,

The non-saponifiable matter is then subjected to high vacuum, unobstructed path distillation. Since the distillate from such high vacuum disand the constant yield liquid is obtained as a distillate or fraction of increased bulk which quickly drains from the condensing surface. Due to the fact that the volum or weight of the constant yield liquid in'the fraction is substantially constant the volume of the constant yield fraction is easily determined, and by simple arithmetic the volume of the desired distillate can also be determined. The utilization of constant yield liquids is, more fully described in an article by- HickmamfIndustrial 8: Engineering Chemistry, vol. 29 (1937) p. 973.

When the'mixture of non-saponifiable matter and constant yield oil is distilled under high vacuum conditions, a distillate which is a mixture of vitamin D and constant yield oil is obtained. Thus each fraction has an appreciable volume and due to its liquid condition, rapidly flows from the still. Sharp fractionating or segregation is thus possible.

Instead of using the constant yield oil expedient, other methods of preventing intermixture may be employed. For instance, the condensing surface can be scraped or wiped with a blade during the course of the distillation. This would effectively remove condensate as rapidly as formed. We also contemplate removing the fraction from the condensing surface by circulating thereover a liquid which dissolves the fraction and rapidly remove it from the still. If

desired, a liquid which is a solvent for the distillate at room or slightly elevated temperature, but a non-solvent at lower temperatures, may be employed to assist in the removal of the vitamin D from the solvent subsequent to the distillation. Such procedure is described in detail in U. S. Patent 2,126,467, granted K. C. D. Hickman on August 9, 1938.

During the distillation six forms of vitamin D will distill at progressively elevated tempera.- tures. The lowest boiling form has a maximum distillation or elimination point of approximately 105. The other five forms exhibit maximum distillation temperatures of approximately 120; 135; and 160; 170; and 185 C. Each of these fractions exhibit a distillation maxima which necessarily indicates the presence of a distinct antirrachitic substance. The lowest boiling form, 1. e. that distilling in maximum amounts at 105 has been tested biologically and found to be abnormally low referred to cod liver oil as standard, to chickens on a rat basis. This substance has been tested biologically and is 25-50 times as potent on a rat basis as calciferol on chickens. It is clear that the vitamins described herein have distinct biological activity and characteristics from that of the mixture in which they have heretofore been prepared.

An alternative procedure to that described above is to first subject the fish oil to high vacuum, unobstructed path distillation to separate the vitamin D content thereof as a concentrate. This concentrate can then be saponified as described and distilled. This procedure has the advantage that saponification of th entire oil is avoided. Obviously, this is desirable since the oil has value and the alkali required to completely saponify is costly.

When the fish oil is directly fractionated, the different forms of vitamin D will be obtained as esters which will respectively distill in maximum amounts at approximately 190; 205; 215; 225;

235 and 255 C. The acids forming the esters are mainly those containing 16 to 20 carbon atoms so that only slight intermixture or overlapping will result.

High vacuum,unobstructed-path distillation is well known in the art. It is that type of distillationwherein the vapor molecules are condensed upon'a condensing surface which is separated from the heated distilland by substantially unobstructed space. It is common to have the heated distilland and the condensing surface separated by a relatively short distance such as to 12 inches, and particularly about 1 to 3 inches. When such distances are used the distillation is known as high vacuum, short path distillation. When the distance between the film and the condensing surface is less than the average distance which a vitamin molecule travels before colliding with a residual gas molecule, the distillation is known as molecular distillation. All of the above types of distillation can be employed for accomplishing the objects of our invention and are to be understood as included within the meaning of the expression high vacuum unobstructed path distillation. Pressures employed should be below 1 mm. and preferably below .1 mm., such as for instance about .01 to .001 mm.

Although we have described our invention. in connection with the isolation of these multiple forms of vitamin D from cod-liver oil, we have found that they occur in fish oils in general, although in different proportions from one oil to another. We, therefore, contemplate applying the principles of our invention to such fish oils. Examples of such oils are tuna, sea bass, halibut, spearfish, barracuda, etc. oils.

E sample 250 grams of KOH were dissolved in 250 grams of water and made up to two liters with 95% ethyl alcohol. 1100 grams of cod liver oil were dissolved in the solution and the resultant mixture kept at about 60 C. forone hour with stirring. It was then cooled down, 8 liters of water was added and the soap mixture then extracted three times with moist ethyl ether. Seven liters were used the first time and two liters for each successive time. The other extracts were combined and washed with water until the washings showed no pink color with phenolphthalein. The ether solution was then dried over night over anhydrous sodium sulfate in an ice box and in an atmosphere of C02. The ether was then removed under reduced pressure and the residue dissolved in methyl alcohol. 0n chilling, a good portion of sterols was precipitated. This was filtered off and the alcohol removed by reduced pressure. The residue was dissolved in cc, of a glyceride constant yield oil and 100 cc. of fish oil molecular distillation residue (components of fish oil which are non-volatile at- 260 C. under molecular distillation conditions). This mixture was distilled in a cyclic molecular still at a pressure of 4 microns. Fractions of about five degree range were withdrawn at and C.

Calciferol has been found to have a distillation maximum of approximately 147 C. The

lowest and highest distilling substances described herein have maximum elimination temperatures of 105 and 185 respectively. Since we have established that every 4-5 change corresponds to one carbon atom it is evident that the 105 antirachitic substance has approximately 8 carbon atoms less than calciferol while the 185 substance has approximately 7 carbon atoms more than calciferol.

The foregoing information obtained. by the 4-5" changes in boiling points or elimination maxima is based upon experimental work with a variety of substances. The results of some of this work are given in an article by us in Industrial 8: Engineering Chemistry," vol. 30 (1938), p. 800 and 801. Reference to this article will show that di-methyl; di-ethyl, di-propinol, di-butyl and di-amyl derivatives of amino-anthraquinone were distilled and it was found that the increase in carbon atoms in each case resulted in an increase in maximum distillation temperature of approximately 4-5 C.

What we claim is:

1. The distillation process whereby a plurality of antirachitic substances contained in a. fish oil can be separated and segregated therefrom, which process comprises in combination converting the antirachitic substances into, free alcohols by saponifying the fish oil, separating the non-saponifiable matter from the saponification mixture, subjecting it to high vacuum, unobstructed path distillation and separating fractions containing different antirachitic substances at approximately 105, 120, 135, 160, 170, and 185 C.

2. The distillation process whereby a plurality of antirachitic substances contained in a fish oil may be segregated, which process comprises in combination subjecting the fish oil to high vacuum, unobstructed path distillation to separate a distillate containing vitamin D in concentrated form, subjecting this distillate to saponification, separating the non-saponifiable matter from the saponification mixture, subjecting it to high vacuum unobstructed path distillation and separating a plurality of fractions containing separate and distinct forms of 'antirachitic substances at approximately 105; 120; 135; 160; 170 and 185 C.

3. As a new composition of matter an antirachitic substance which has a lower potency on chickens, on a rat basis than vitamin D prepared by saponiflcation of a fish oil, but being about 25 to 50 times as potent as calciferol, which new composition of matter has approximately 8 carbon atoms less than calciferol and is obtained by subjecting a fish oil to saponification, separating the non-saponifiable matter,

3 subjecting the non-saponifiabie matter to high vacuum, unobstructed path distillation, and separating an antirachitic substance which distills 'in maximum amounts at a temperature of approximately 105 C.

' KENNETH C. D. HICKMAN.

EDWARD LE B. GRAY. 

